in vitro comparative metabolism studies to identify metabolites … · 2019-04-05 · for chemical...
TRANSCRIPT
In vitro comparative metabolism studies to
identify metabolites using microsomes:
standards and criteria for acceptability and
interpretation
Khaled Abass, Ph.D., ERT
Faculty of Medicine, University of Oulu, Finland
in vitro comparative metabolism studies in regulatory pesticide risk assessment, EFSA, 15-16 November 2018
Outlines
- Introduction
- Case studies (comparative qualitative and quantitative metabolism)
- Pesticide-CYP interactions
- Conclusion
Introduction
Enzyme sources Availability Advantages Disadvantages
Microsomes Relatively good, from transplantations or commercial sources
Major Phase I enzymes. Inexpensive technique. Easy storage. Study of species-specific metabolic profile.
Cellular and organ architecturelost, Cofactor addition necessary, Lack of active uptake and transport
Complexity Easy applicable Ethically acceptable Resemblance of in vivo
Recombinant enzymes
Microsomes
S9 fraction
Cell lines
Primary hepatocytes
Liver slices
In vivo animal model
Human
Models in order of in vivo resemblance.
Case studies-qualitative and quantitative comparative metabolism
Abass et al. Pestic. Biochem. Physiol. 87 (2007)
- Profenofos (Class: Organophosphorothiolate) - Diuron (Class: Phenyl urea)
Cl
Cl
NH C NCH3
CH3
O
Br
OP
O
OS
Cl
- Carbosulfan (Class: Carbamates)
Abass et al. Drug Metab. Dispos. 35 (2007) Abass Pestic. Biochem. Physiol. 107 (2013)
Abass et al. Chem. Biol. Interact. 181 (2009)
Abass et al. Chem. Biol. Interact. 185 (2010)
- Benfuracarb (Class: Carbamates)Abass et al. Toxicology Letters 224 (2014), pp. 209-299
Abass et al. Toxicology Letters 224 (2014), pp. 300-310
OCH3
CH3
O C
N S
O
CH3 N
CH2
CH2
CH2
CH3
CH2
CH2CH2
CH3
OCH3
CH3
O
N S
O
CH3 N
H3C(H2C)3
(CH2)3CH3
1-Profenofos
Overall scheme of profenofos metabolism in human, mouse, and rat hepatic microsomes and exact
masses of metabolites Abass et al. Pestic. Biochem. Physiol. 87 (2007)
1-Profenofos
Kinetic parameters for the formation of profenofos metabolites by human,
mouse and rat hepatic microsomes
Metabolites CLintµl / (mg protein * min)
Bioactivation/
hydroxylationIn vitro species differences
HumanLM
Desthiopropylation
Hydroxylation
27.9
0.382
MouseLM
Desthiopropylation
Hydroxylation
37.5
2.714
1.3
9.0
RatLM
Desthiopropylation
Hydroxylation
8.8
1.95
0.3
6.3
Interspecies differences represents animal to human fold differences in in vitro toxicokinetics
Abass et al. Pestic. Biochem. Physiol. 87 (2007)
Abass et al. Drug Metab. Dispos. 35 (2007) Abass Pestic. Biochem. Physiol. 107 (2013)
2-Diuron
The overall scheme of
the diuron metabolites
detected in
postmortem and
hospitalized cases. In
addition to in vitro
metabolism by seven mammalian LM. postmortem case (full line)
hospitalized case (discontinuous line)
2-Diuron
Kinetic parameters of N-demethyldiuron formations obtained with different
mammalian liver microsomes
Clint µl / (mg protein * min) In vitro species differences
HumanLM 174.2
RatLM 74.7 0.43
MouseLM 214.0 1.23
DogLM 401.3 2.30
MonkeyLM 327.2 1.88
MiniPigLM 159.7 0.92
RabbitLM 314.1 1.80
Interspecies differences represents animal to human fold differences in in vitro toxicokinetics
Abass et al. Drug Metab. Dispos. 35 (2007) Abass Pestic. Biochem. Physiol. 107 (2013)
3-Carbosulfan
The overall in vitro scheme of
carbosulfan metabolism in mammalian
liver microsomes
Abass et al. Chem. Biol. Interact. 181 (2009)Abass et al. Chem. Biol. Interact. 185 (2010)
ratLM x
ratLM x
mouseLM x
rabbitLM
DogLM x
Kinetic parameters of the carbofuran- metabolic pathway obtained with
different mammalian liver microsomesa
Clint µl / (mg protein * min) In vitro species differences
HumanLM 454.9
RatLM 326.6 0.72
MouseLM 253.2 0.56
DogLM 223.9 0.49
RabbitLM 335.7 0.74
MinipigLM 471.4 1.03
MonkeyLM 450.8 0.99
Abass et al. Chem. Biol. Interact. 181 (2009)Abass et al. Chem. Biol. Interact. 185 (2010)
a Sums of all the metabolites of the carbofuran pathway were used for the calculation of kinetic parametersb Interspecies differences represents animal to human fold differences in toxicokinetics
3-Carbosulfan
4-Benfuracarb
The overall in vitro scheme of
benfuracarb metabolites
detected in mammalian liver
microsomes. The percentage of metabolite formation in HLM
and the contribution of hrCYP
are shown.
Abass et al. Toxicology Letters 224 (2014), pp. 209-299Abass et al. Toxicology Letters 224 (2014), pp. 300-310
DogLM x
DogLM x
Rat in vivo x
Kinetic parameters of the carbofuran- metabolic pathway obtained with different
mammalian liver microsomesa
Clint µl / (mg protein * min) In vitro species differences
HumanLM 99.95
RatLM 253.7 2.5
MouseLM 255.2 2.5
DogLM 145.8 1.4
RabbitLM 134.7 1.3
MinipigLM 268.9 2.7
MonkeyLM 143.9 1.4
a Sums of all the metabolites of the carbofuran pathway were used for the calculation of kinetic parametersb Interspecies differences represents animal to human fold differences in in vitro toxicokinetics
Abass et al. Toxicology Letters 224 (2014), pp. 209-299Abass et al. Toxicology Letters 224 (2014), pp. 300-310
4-Benfuracarb
Qualitative Differences
Profenofos Diuron Carbosulfan Benfuracarb
HumanLM - - In vivo vs in vitro - -
RatLM - - 3-Keto-7-PhCF ND
3-OH-7-PhCF ND
3-Keto-CF ND in vivo
MouseLM - - 3-OH-7-PhCF ND -
DogLM - Carbofuran ND 3-Keto-7-PhCF ND
3-OH-7-PhCF ND
RabbitLM - 7-PhCF specific metab. -
MinipigLM - - -
MonkeyLM - - -
Quantitative Differences
Profenofos Diuron Carbosulfan Benfuracarb
CLint CLH CLint CLH CLint CLH CLint CLH
HumanLM
RatLM 1.3 3.3 0.43 2.3 0.72 3.1 2.5 4.1
MouseLM 0.3 4.1 1.23 3.9 0.56 3.5 2.5 4.8
DogLM 2.30 2.2 0.49 1.7 1.4 2.1
RabbitLM 1.88 2.3 0.74 2.0 1.3 2.3
MinipigLM 0.92 1.7 1.03 1.9 2.7 2.4
MonkeyLM 1.80 2.7 0.99 2.5 1.4 2.3
Animal to human quantitative differences (fold) in toxicokinetic for the active chemical moieties The Extrapolated CLH in animals were divided by the CLH in humans
in vitro and in vivo extrapolated values for animal to human differences (fold) in
toxicokinetic for the active chemical moieties
Abass Pestic. Biochem. Physiol. 107 (2013)
Each pesticide is ‘‘an individual’’ with its own characteristics regarding toxicokinetics and metabolic pathway
DMA/2
Mouse
Sprague-Dawley
Rat
Beagle
Dog
Human
Caucasian race
Cynomolgus
Monkey
New Zealand
White Rabbit
Göttingen
Minipig
In vitro hepatic model (microsomes)
Metabolic Profile Quantitative analysis In vitro-in vivo extrapolation
Proper selection and interpretation of animal models for toxicological evaluation and chemical risk assessment
Comparative in vitro metabolism studies
Abass et al. Toxicology Letters 224 (2014), pp. 209-299
Limitations
- Results obtained from in vitro test systems are highly dependent on several
technical factors
- The prediction of in vivo metabolic clearance is based on many scaling factors
and physiological measures, which should either be assumed or measured.
- The best predictive value is usually obtained when the substrate concentration
used is within the linear part of the time and protein concentration curves for
substrate depletion or metabolite formation
Comparative in vitro metabolism studies
The percentage of hrCYPs involved in pesticides metabolism. 63 compounds (36 insecticides; 14 fungicides; 10 herbicides; 2 plant growth regulators and a biocide agent) were metabolized at least in part by one or more hrCYP yielded 495 metabolic reactions (restricted to 2011 survey).
Abass et al. 2012, Insecticides, ISBN 979-953-307-667-5, pp. 165-194.
Pesticides-CYP Intercations
mRNA 10 µM /fold induction 50 µM /fold induction
CYP1A2 TCDD 218 (10 nM)
Diuron 9 100
CYP2B6 Phenobarbital 6 (500 µM)
Isoproturon 20 22
Atrazine 11 13
CYP3A4 Rifampicin 20 19
Cypermethrin 6 35
Fenvalerate 17 22
Cyhalothrin 13 28
CYP mRNA levels in human HepaRG after 24 h exposure to tested pesticides.
hPXRhCAR
XREM
24 structurally diverse pesticides
11 hCYP
Xenbioticcomplex
11 hCYP
Abass et al. 2012, Toxicology. 294: 17–26Abass et al. 2013 Toxicol In Vitro. 27(5) 1584-8
Pesticide-CYP Intercations
Abass et al. 2012, Toxicology. 294: 17–26Abass et al. 2013 Toxicol In Vitro. 27(5) 1584-8
mRNA 10 µM /fold induction 50 µM /fold induction
CYP1A2 TCDD 218 (10 nM)(20-fold Mela-OH)
Diuron 9 100-fold (IC50 =3 µM)
CYP2B6 Phenobarbital 6 (500 µM)(3-fold Bup-OH)
Isoproturon 20(7-fold Bup-OH)
22 (7-fold Bup-OH)
Atrazine 11 (3-fold Bup-OH)
13 (4-fold Bup-OH)
CYP3A4 Rifampicin 20(10-fold Tes-6OH)
19(10-fold Tes-6OH)
Cypermethrin 6 35
Fenvalerate 17(3-fold Tes-6OH)
22(10-fold Tes-6OH)
Cyhalothrin 13(5-fold Tes-6OH)
28(8-fold Tes-6OH)
Pesticide-CYP Intercations
Pesticides can induce/inhibit the CYPs
involved in their own metabolism
CYP mRNA levels in human HepaRG after 24 h exposure to tested pesticides.
The observed differences emphasize the importance of using human-based cellular
screening models in comparative metabolism studies
Pesticide-CYP Intercations-Species differences
Nuclear receptor activity was measured by luciferase reporter gene CYP-mRNA induction in HepaRG and in mouse primary hepatocytes
Abass et al. 2012, Toxicology. 294: 17–26
Comparison of the mouse and human CAR/PXR and CYP2B/CYP3A mRNA induction by 24 structurally diverse pesticides
Conclusion
in vitro screening of metabolite profiles and toxicokinetics are desirable for the
proper selection of animal models for toxicological evaluation
- Rate of metabolism
- Spectrum of metabolites produced
- Intrinsic clearance
to include interactions at different biological levels and to maximize the chance
of having all possible metabolites, Living cells, if metabolically competent, would
give metabolite patterns closer to in vivo situation than tissue fractions.
References Abass et al. 2016 Approches to desribe risks and future needs. In Arctic Monitoring and Assessment Programme AMAP 2015, Oslo,
Norway, ISBN: 978-82-7971-093-6.
Abass et al. , 2013 Human variation and CYP enzyme contribution in benfuracarb metabolism in human in vitro hepatic models. Toxicology letters 224 (2), 300-309
Abass et al. 2014 Comparative metabolism of benfuracarb in in vitro mammalian hepatic microsomal model and its implications for chemical risk assessment. - Toxicology letters 224 (2), 290-299.
Pelkonen et al. 2013. How to preserve, induce or incorporate metabolism into the in vitro cellular system, Toxicology in vitro. 27.
Abass K. 2013 From in vitro hepatic metabolic studies towards human health risk assessment: Two case studies of diuron and carbosulfan. - Pesticide Biochemistry and Physiology 107 (2), 258-265
Abass and Pelkonen 2013 The inhibition of major human hepatic cytochrome P450 enzymes by 18 pesticides: comparison of the N-in-one and single substrate approaches. - Toxicology in vitro 27, 1584-1588
Abass et al 2012. Characterization of human cytochrome P450 induction by pesticides. - Toxicology 294 (1), 17-26.
Abass et al. 2009 Metabolism of carbosulfan. I. Species differences in the in vitro biotransformation by mammalian hepatic microsomes including human. - Chemico-Biological interaction 181, 210-219
Abass et al. 2010 Metabolism of carbosulfan II. Human interindividual variability in its in vitro hepatic biotransformation and the identification of the cytochrome P450 isoforms involved. - Chemico-biological interactions 185, 163-173
Abass et al. 2009 Evaluation of the cytochrome P450 inhibition potential of selected pesticides in human hepatic microsomes. -Journal of Environmental Science and Health Part B 44, 553-563
Abass et al. 2007 Characterization of diuron N-demethylation by mammalian hepatic microsomes and cDNA-expressed human cytochrome P450 enzymes. - Drug metabolism and disposition 35, 1634-1641
Abass et al. 2007 In vitro metabolism and interaction of profenofos by human, mouse and rat liver preparations. - Pesticide Biochemistry and Physiology 87, 238-247
Abass et al. 2011 Metabolism of pesticides by human cytochrome P450 enzyems in vitro - a survey. Insecticides - - ISBN 979-953-307-667-5, pp. 165-194
Thank you
Arctic Health; University of Oulu, Finland